Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves

Growth Hormone Secretagogues Protect Mouse Cardiomyocytes from in vitro Ischemia/Reperfusion Injury through Regulation of Intracellular Calcium

Background: Ischemic heart disease is a leading cause of mortality. To study this disease, ischemia/reperfusion (I/R) models are widely used to mimic the process of transient blockage and subsequent recovery of cardiac coronary blood supply. We aimed to determine whether the presence of the growth hormone secretagogues, ghrelin and hexarelin, would protect/improve the function of heart from I/R injury and to examine the underlying mechanisms. Methodology/Principal Findings: Isolated hearts from adult male mice underwent 20 min global ischemia and 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nM) or hexarelin (1 nM) was introduced into the perfusion system either 10 min before or after ischemia, termed pre- and post-treatments. In freshly isolated cardiomyocytes from these hearts, single cell shortening, intracellular calcium ([Ca ] ) transients and caffeine-releasable sarcoplasmic reticulum (SR) Ca were measured. In addition, RT-PCR and Western blots were used to examine the expression level of GHS receptor type 1a (GHS-R1a), and phosphorylated phospholamban (p-PLB), respectively. Ghrelin and hexarelin pre- or post-treatments prevented the significant reduction in the cell shortening, [Ca ] transient amplitude and caffeine-releasable SR Ca content after I/R through recovery of p-PLB. GHS-R1a antagonists, [D-Lys3]-GHRP-6 (200 nM) and BIM28163 (100 nM), completely blocked the effects of GHS on both cell shortening and [Ca ] transients. Conclusion/Significance: Through activation of GHS-R1a, ghrelin and hexarelin produced a positive inotropic effect on ischemic cardiomyocytes and protected them from I/R injury probably by protecting or recovering p-PLB (and therefore SR Ca content) to allow the maintenance or recovery of normal cardiac contractility. These observations provide supporting evidence for the potential therapeutic application of ghrelin and hexarelin in patients with cardiac I/R injury

Modeling CICR in rat ventricular myocytes: voltage clamp studies

<p>Abstract</p> <p>Background</p> <p>The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect <it>Ca</it>²⁺loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR <it>Ca</it>²⁺release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic <it>Ca</it>²⁺concentration ([<it>Ca</it>²⁺]<it>_myo</it>).</p> <p>Methods</p> <p>The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed <it>Ca</it>²⁺channels (trigger-channel and release-channel). It releases <it>Ca</it>²⁺flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[<it>Ca</it>²⁺]<it>_myo</it>.</p> <p>Results</p> <p>Our model reproduces measured VC data published by several laboratories, and generates graded <it>Ca</it>²⁺release at high <it>Ca</it>²⁺gain in a homeostatically-controlled environment where [<it>Ca</it>²⁺]<it>_myo</it>is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR <it>Ca</it>²⁺release, its activation by trigger <it>Ca</it>²⁺, and its refractory characteristics mediated by the luminal SR <it>Ca</it>²⁺sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence <it>Ca</it>²⁺homeostasis.</p> <p>Conclusions</p> <p>We examine the role of the above <it>Ca</it>²⁺regulating mechanisms in handling various types of induced disturbances in <it>Ca</it>²⁺levels by quantifying cellular <it>Ca</it>²⁺balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.</p

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

8. SEEN Workshop. Praha‚ Czech Republic 2-5 February 2006

Program a abstrakta 8. mezinárodního workshopu South East European Bird Migration Network (SEEN). Na CD-ROM jsou panely prezentace České společnosti ornitologické